Field of the Invention
The field of the invention relates to determination of the profile of unbound free fatty acids (FFAu) in biological samples, such as human and animal blood specimens and plant and animal oils, by measuring the fluorescence response of sets of different fluorescently labeled fatty acid binding proteins (probes) that undergo a change in fluorescence ratio at 2 wavelengths upon binding an FFAu. The field also relates to the use of these profiles in human and animal disease, in basic research, in drug development and in industrial uses.
Description of the Related Art
For purposes of the present disclosure, “fatty acids”, are non-esterified carboxylated alkyl chains of 1-30 carbons atoms which may exist as neutral (e.g. protonated, sodium or potassium salt) or ionic species, depending upon the pH and conditions of the aqueous media. “Free fatty acids (FFA)” are equivalent to fatty acids and both terms refer to the totality of FFA including those in aqueous solution as monomers plus those that are not in solution (for example bound to other macromolecules (proteins, membranes), cells, oil droplets, or part of an aggregate of FFA (micelles, soaps and other more complex aggregates). FFA present as monomers in aqueous solution (either charged or neutral) are referred to as “unbound free fatty acids (FFAu)”. For purposes of the present disclosure, FFA are molecules whose molecular weight ranges from about 70 to about 500 Da and FFAu are these molecules in aqueous solution.
For the purposes of the present disclosure, the term “lipid” is taken to have its usual and customary meaning and defines a chemical compound which is most soluble in an organic solvent but has some level of solubility in the aqueous phase (the fraction that is unbound). FFAs are a type of lipid and FFAu are that fraction of FFA that are soluble in the aqueous phase. Accordingly, a “lipid-binding protein” includes any protein capable of binding a lipid as lipid is defined herein.
Levels of unbound FFA provide information diagnostic of health and disease when measured in appropriate human or animal fluids (Richieri G V and Kleinfeld A M (1995) J Lipid Res 36: 229-240, Kleinfeld A M, et al. (1996) Am J Cardiol 78: 1350-1354, Cantor W J et al, (2008) J Invasive Cardiol 20: 186-188., Bhardwaj A, et al (2011). Am Heart J 162: 276-282, Hegyi T, et al (2013). Neonatology 104: 184-187, 2013. Huber A H, et al (2013) Am J Cardiol. 113:279-284. FFAu levels provide information essential to fundamental biology and have important applications in drug development and industry. It is increasingly apparent that determination of the unbound (a.k.a ‘aqueous phase’ or ‘free’) concentration of such molecules provides critical information about physiologic homeostasis. Many FFA are hydrophobic molecules with low aqueous solubility and unbound concentrations that are much lower than their “total” concentration, where the bulk of the “total” may be bound to proteins or cells. In biological fluids the concentration of the unbound FFA is often regulated to maintain a relatively constant unbound concentration under normal physiologic conditions. Much of this regulation occurs through the interaction of the molecules with a carrier protein such as for example, albumin. Thus most of the physiologically important medium and long chain FFA are generally bound to albumin, or other carriers. However a small fraction of the molecules may dissociate (and rebind to) from the albumin into the aqueous phase and these are the unbound molecules.
Intracellular lipid binding proteins (iLBP) are a family of low-molecular weight single chain polypeptides all of whom have similar 3 dimensional atomic structures. There are four recognized subfamilies. Subfamily I contains proteins specific for vitamin A derivatives such as retinoic acid and retinol. Subfamily II contains proteins with specificities for bile acids, eiconsanoids, and heme. Subfamily III contains intestinal type fatty acid binding proteins (FABPs) and Subfamily IV contains all other types of fatty acid binding protein (Haunerland, et al. (2004) Progress in Lipid Research vol. 43: 328-349). The entire family is characterized by a common 3-dimensional fold. Ligand binding properties of the different subfamilies overlap considerably. The wild type proteins of subfamily I (Richieri et al (2000) Biochemistry 39:7197-7204) and subfamily II both bind fatty acids as well as their native ligands. Moreover, single amino acid substitutions are able to interconvert the ligand binding properties of proteins of subfamilies I and II (Jakoby et al (1993) Biochemistry 32:872-878).